Title page for ETD etd-09252008-093620

Document Type Master's Dissertation
Author Koot, Dwayne Jonathan
Email dwaynekoot@hotmail.com
URN etd-09252008-093620
Document Title An investigation into the antineoplastic potential of Bridelia micrantha constituents
Degree MSc
Department Pharmacology
Advisor Name Title
Prof C E Medlen Co-Supervisor
Dr A D Cromarty Supervisor
  • chromatography
  • antineoplastic
  • bioassay-guided fractionation
  • botanicals
  • Bridelia micrantha
  • cancer
  • chemotaxonomy
  • ethnobotanical
  • phytochemical
  • secondary plant metabolites
Date 2007-11-24
Availability restricted
The fight against cancer is on going. This study provides a brief overview of the current status of cancer. Natural products as a source of clinically relevant, biologically active compounds, is the central theme of this study. Special reference has been made to antineoplastic (cancer-combating) compounds derived from botanicals.

Bridelia micrantha is a multipurpose tree used crudely by several African cultures for the treatment of a variety of ailments. Suggestive cytotoxic results attained from a preliminary toxicological screening along with the knowledge that the genus Bridelia has proven to be a source of novel cytotoxic aryltetralin lignan glycosides (structurally related to podophyllotoxin), served as motivation for undertaking this study.

The in vitro antineoplastic potential of several different solvent extracts of B. micrantha (bark) has been investigated. Five out of the six primary extracts displayed impressive cytotoxicity (IC50<10g/ml) towards three out of the four tested neoplastic cell lines. Indicative of good antineoplastic potential, these same extracts did not inhibit the growth of primary porcine hepatocytes and furthermore significantly (P<0.05) stimulated the proliferation of primary human lymphocytes at comparable, physiologically relevant concentrations. The presence of numerous structurally distinct cytotoxic compounds was shown by statistical comparisons (2way ANOVA and Bonferroni multiple comparison post-tests) of the IC50< values produced by each of the primary extracts on each of the tested neoplastic cell cultures.

The manner of cell death induction and points of cell cycle inhibition induced by the most promising of the primary extracts (H2<0SIS< - lyophilised aqueous extract, soluble in EtOH) at a fixed concentration of 10g/ml, was assessed flow cytometrically using COLO 320DM (human colorectal adenocarcinoma) cells. After 24hours incubation, extract H20S1S was shown to induce significant apoptosis (P<0.005) relative to appropriate controls. During that same period, the progression of the cell cycle was seemingly not hindered.

The active constituent/s (AC) within extract H2<0SIS< were sought through bioassay-guided fractionation using the HeLa (human cervical epitheloid carcinoma) cell line as a monitor of bioactivity. Direct-infusion ESI-MS, HPLC/DAD/MS and TLC were used as monitors of fraction constituency. Fractions eluted from C18 and ion exchange sorbents were shown to possess decreased biological activity owing to incomplete distribution of the analytes and/or the separation of numerous compounds, acting additively. Biological activity was enhanced through solvent-solvent partitioning between butanol (BuOH) and water (H2 Attempts to further enrich the AC using normal phase preparative thin layer chromatography (pTLC) were met with a fair degree of separation. These fractions could unfortunately not be assayed due to appreciable solubilization of silica during analyte recovery and the resultant problems with accurate quantitation. Column chromatography employing silica as the stationary phase appears to be an attractive next fractionation step.

As yet the constituent/s responsible for the impressive antineoplastic activity have not been isolated nor structurally identified. Chemical (chromogenic) tests conducted on developed TLC plates do however suggest the possible presence of aryltetralin lignans. The results reported and discussed are of clinical relevance and certainly indicate that further investigation is required.

University of Pretoria 2007


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